Lampreys swim by rhythmic lateral undulations, and the neuronal analog of this behavior (fictive swimming) can be recorded from the lamprey spinal cord in vitro. Analysis of the cellular basis of fictive swimming by intracellular recording techniques has made the lamprey an important model system in which to understand rhythmic behaviors and their cellular mechanisms, a major goal of neurobiology. It is hoped that understanding the cellular basis of spinal cord function in a lower vertebrate will yield general insights into spinal organization and may assist in the rational design of therapies for humans suffering from spinal cord injury or disease. A number of spinal interneurons involved in fictive swimming have been identified physiologically, but their synaptic interactions are only partially known. In addition, other swim-related interneurons await physiological analysis, in part because of their small size and low numbers. The proposed project will exploit the long-projecting axons of intersegmental interneurons to label the cell bodies with fluorescent tracers for visually-guided impalement with intracellular electrodes. Seven fluorescent tracers will be tested for their effectiveness at retrograde labelling of intersegmental interneurons, and an in vitro preparation of the spinal cord and notochord will be used to determine if fluorescent-labelled cells can be visualized and recorded intracellularly. Following establishment of the labelling technique, it will be applied to characterize the cellular properties and synaptic interactions of spinal neurons with ascending axons. Evidence from extracellular recordings indicates that these neurons are important mediators of intersegmental coordination, but intracellular recordings from them have not been reported. Study of the ascending neurons is part of a long-term project to identify the synaptic interactions that are important for fictive swimming, and also to examine the modulation of those synapses by endogenous amines and peptides.